PROJECT SUMMARY Zika virus (ZIKV) is an enveloped flavivirus transmitted by Aedes mosquitos which leads to an asymptomatic or mild dengue-like disease. A large ZIKV outbreak started in Brazil in early 2015, recently spreading to more than 25 countries in the Americas. Alarmingly, the ZIKV Brazilian outbreak has been linked to thousands of microcephaly cases in babies born to infected mothers, leading the WHO to recently declare a Public Health Emergency of International Concern. Thus, control efforts are desperately needed to contain the outbreaks and avoid further spread to other countries. No ZIKV vaccines have been developed, although vaccines for the related dengue virus (DENV) based on the Membrane (M) and Envelope (E) proteins, which induce protective immunity, are in advanced clinical trials, and one (Dengvaxia) was just approved in Mexico, Brazil, and the Philippines. Vaccinia virus (VACV) was used to eradicate smallpox, a disease caused by variola virus, a related poxvirus. VACV has also been successfully used as a replication-competent or -defective viral vector for the development of effective human and animal vaccines, as it elicits strong and long-lasting humoral and cell-mediated immune (CMI) responses to heterologous antigens expressed in its genome. We recently generated VACV vectors with a built-in safety mechanism that replicate only in the presence of tetracycline antibiotics. When administered as a vaccine (in the absence of antibiotics), the vector does not replicate but retains its immunogenicity, and therefore is safer for human use. Conveniently, the vector can be propagated in cell culture at high titers in the presence of tetracyclines, unlike other replication-defective VACV-based vectors such as modified vaccinia Ankara (MVA). More recently, we developed a novel method to generate and purify recombinant VACV vaccines without the use of selection markers in as little as one week, a process that normally takes months. Our goal is to use this method to rapidly generate recombinant VACV vaccines expressing the ZIKV E and M proteins and to test their immunogenicity, safety, and efficacy in mice. In Aim 1 we will rapidly generate and characterize replication-defective VACV vaccine candidates expressing the ZIKV E protein (VACV-ZIKVs). A number of ZIKV E and M-E gene constructs were designed for expression of E alone or as virus-like particles (VLPs) to maximize induction of serum neutralizing (SN) antibodies. In Aim 2 we will test the immunogenicity (SN titers and CMI responses) and safety of VACV-ZIKV vaccine candidates in mice. Concurrently, in Aim 3 we will develop a ZIKV mouse challenge model based on current models for the related DENV to test the efficacy of our VACV-ZIKV vaccine candidates. Our goal is to use our accelerated VACV platform to rapidly select the best antigen strategy that leads to high levels of SN antibody production, CMI responses, and protection in mice.